Cardiomyocyte Differentiation Issue

hiPSC Culture Discussion
6

Hi @hwidyastuti

We do have a protocol for troponin-T staining of fixed cells (fixed in 4%PFA/PBS for 10 min). The steps are as follows:

Flow Cytometry Prep

  • In round bottom 96 well plate, plate 2 wells per sample, 80-100ul cell suspension/well.
  • Spin down at 200g, 3min, 25C (radius=15.1)
  • Flick off supernatant
  • Resuspend pellets in 99.5ul BD Perm/Wash + 0.5ul anti-Troponin T AF647 or isotype control
  • Incubate at room temperature in dark for 30min
  • Add 100ul BD Perm/Wash to wells
  • Spin down at 200g, 3min, 25C
  • Flick off supernatant
  • Add 200ul BD Perm/Wash, pipette gently 5x. Spin down at 200g, 3min, 25C. Flick off sup’t.
  • Add 200ul PBS/5%FBS, pipette gently 5x. Spin down at 200g, 3min, 25C. Flick off sup’t.
  • Add 200ul PBS/5%FBS, pipette gently 5x. Spin down at 200g, 3min, 25C. Flick off sup’t.*
  • Resuspend pellets in 200ul PBS/5%FBS+DAPI(1:1000), filter with 35um mesh cap into FACS tubes for acquisition.

We use the BD FACS AriaIII for sorting. Please let us know if you have further questions! Happy to help.